Method for producing methane by means of aerobic co-culture of anaerobic micro-organisms

ABSTRACT

The present invention relates to the biological production of methane (Biogas) by co-culture in an aerobic atmosphere of a methanogenic bacterium and of an anaerobic bacterium capable of producing hydrogen, in a culture medium comprising or being supplemented with carbohydrate compound(s), notably starch and/or sugars, and supplemented with antioxidant compound(s).

The present invention relates to a method for producing methane gas(CH₄) by microbial culture under aerobic conditions of anaerobicmicroorganisms.

Methane produced biologically has been considered until recently as onlybeing produced in an anaerobic way, i.e. without oxygen, and usually inthe presence of hydrogen, by anaerobic fermentation of a certain numberof organic wastes.

In FR 2 537 992, CH₄ gas is produced by fermentation and anaerobicdegradation of organic wastes. For this, hydrogen is injected whichreacts with the produced CO₂ in order to form CH₄ and thus reduce theCO₂ content in a mixture with CH₄. Ten to 5,000 liters of H₂ gas per m³of produced CH₄ are required.

In FR 2 601 690, a thermophilic methanogen Methanococcusthermolitotrophicus is used with a supply of H₂/CO₂, the cultivationbeing accomplished in a medium essentially containing a source ofnitrogen and a source of salts which may be assimilated at 110° C. underanaerobic conditions.

Recent research work, since 2006 (1), have shown that in the sea it waspossible to detect methane in an aerobic medium exclusively afterenrichment with methylphosphonate, with one or several microorganismsfixing the hydrogen (Karl D M, Beversdorf L, Bjorkman K M, Church M J,Martinez A, DeLong E F. Aerobic production of methane in the sea. NatGeosci 2008; 1:473-8).

In GB 2,107,735, a fermentation method is described for producingmethane gas comprising the co-culture of a Methanobacterium strain andof a bacteria Clostridium bifermentans under anaerobic conditions underan atmosphere containing hydrogen and carbon dioxide (CO₂).

Moreover, natural methane is produced in the intestine under anaerobicconditions under the activity of methanogenic microorganisms. In thedigestive tract, the methane is produced under the action of Archaemethanogenic microorganisms called in particular Methanobrevibactersmithii or Methanomassilicoccus luminyensis, which produce methane fromhydrogen molecules (H₂) produced by the fermentation of sugars withanaerobic bacteria of the digestive tract, in particular by Bacteroides,in particular by Bacteroides thetaiotaomicron.

The object of the present invention is to provide a novelbiotechnological method for producing methane under aerobic conditions.

The inventors surprisingly discovered that it was possible to allow thecultivation of anaerobic bacteria by associating with the culturemedium, an antioxidant compound while maintaining the culture ofmethanogenic archaea.

More specifically, the inventors observed that the addition ofantioxidant compounds in a culture medium under an aerobic atmospherewith an anaerobic bacterium capable of producing hydrogen, also allowedcultivation of a methanogenic archaea, because of the production ofhydrogen by said anaerobic bacterium. They were able to measure theproduction of hydrogen in the presence of said anaerobic bacterium andthe production of methane by said archaea, made under aerobicconditions, by the association of two anaerobic microorganisms, notablyMethanobrevibacter smithii or Methanomassiliicoccus luminyensis andBacteroides thetaiotaomicron. Thus, producing co-culture chambers withBacteroides thetaiotaomicron on the one hand or another anaerobicbacterium, and on the other hand Methanobrevibacter smithii orMethanomassiliicoccus luminyensis, or another methanogenic organismgives the possibility of providing a significant amount of methane.

The present invention therefore essentially consists in the biologicalproduction of methane (Biogas) by an association of a methanogenicbacterium and of an anaerobic bacterium being cultivated under aerobicconditions in a medium rich in antioxidant components and containing ahydrocarbon source.

To do this, the present invention provides a method for producingmethane gas in a reactor by co-culture under an aerobic atmosphere,preferably of ambient air, of at least:

-   -   a first microorganism consisting in an anaerobic bacterium able        to produce hydrogen by fermentation in the presence of a        substrate and/or a culture medium comprising or supplemented        with carbohydrate(s), notably starch and/or sugars, and    -   a second microorganism consisting in a methanogenic Archaea able        to produce methane from hydrogen and from a substrate and/or        culture medium comprising or supplemented with hydrocarbon        compound(s), notably starch and/or sugars, and    -   an organic and mineral substrate comprising components of        culture media able to allow cultivation of both said first and        second anaerobic microorganisms, said culture medium comprising        or being supplemented with carbohydrate(s), notably starch        and/or sugars, and further being supplemented with antioxidant        compound(s).

More particularly, the other cultivation conditions, notablytemperature, are suitable conditions for the cultivation of saidmicroorganisms. In particular, the temperature has to be increased inorder to incubate if necessary to a temperature promoting growth of saidmicroorganisms, notably at least 30° C. or even 37° C.

It is understood that the method according to the present invention doesnot require the supply of external hydrogen and that the co-culturereactor comprises means for recovering the produced methane gas.

Preferably, the cultivation of said first microorganism is achievedfirst of all in said substrate and said second microorganism isintroduced after said first microorganism has already producedfermentation products and hydrogen.

By “antioxidant compound”, is meant here a compound with an antioxidantproperty, i.e. it reduces or prevents oxidation of other substances,notably in this case, other substances involved in the method of theinvention under the conditions of the method according to the invention.

More particularly, said antioxidant compound is preferably selected fromamong ascorbic acid, uric acid and glutathione(γ-L-Glutamyl-L-cysteinylglycine). Ascorbic acid and uric acid arepreferred since they are capable at specific doses of allowingcultivation at a higher oxygen level.

Still more particularly, said antioxidant compound is applied at aconcentration of 1 μg/ml to 2 mg/ml, or in a molar concentration from10⁻⁶ M to 10⁻² M, preferably at least 1 g/l.

Preferably, said medium comprises a pH-regulating buffer substance foradjusting the pH from 7 to 7.5.

More particularly, said Archaea is an Archaea selected from thefollowing Archae: Methanobrevibacter, Methanosphaera,Methanomassiliicoccus, Methanobacterium, Methanococcus and Methanosaeta.

More particularly, said Archaea is a Archaea selected from the followingArchae: Methanobrevibacter smithii, Methanobrevibacter orails;Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis,Methanobacterium beijingense and Methanosaeta concilii.

Still more particularly, said Archea is a Methanobrevibactera smithii orMethanomassiliicoccus luminyensis.

Strict anaerobic bacteria, i.e. they are not capable of being cultivatedin the presence of oxygen or in concentrations of less than 1%, morecommonly less than 0.1%, ideally 0%. From among strict anaerobicbacteria, more particularly mention is made of extracellular bacteria,i.e. bacteria which can only live on the exterior of cells.

More particularly, said anaerobic bacterium able to produce hydrogen isselected from bacteria from the Actinobacteria and Bacteroidetesfamilies.

Preferably, said anaerobic bacterium is of the Bacteroides genus, thesebacteria are in particular known for digesting starch while producinghydrogen.

More particularly, said anaerobic bacterium is Bacteroidesthetaiotaomicron.

The bacteria Lactococcus lactis is also mentioned as an Actinobacteria.

More particularly, said culture medium comprises components which arefound in the culture basis media able to cultivate an archaea or ananaerobic bacterium, comprising at least:

-   -   several carbon sources,    -   a phosphorus source, preferably a phosphate salt,    -   a source of nitrogen, preferably an ammonium salt,    -   at least one salt of metal selected from among K, Mg, Na, Ca,        preferably NaCl.

More particularly, said culture medium is an acellular medium, isselected from among an axenic medium consisting of chemical orbiological substances defined qualitatively and quantitatively, and anacellular medium comprising an extract of milled or lyzed material frompluricellular tissue.

More particularly, said culture medium is a conventional acellularmedium of an anaerobic bacterium, preferably a medium comprisingcomponents selected from among an extract of pluricellular tissue milledmaterial or lyzed material, an enzymatic digested material, notably anenzymatic digested material of casein, soy and/or animal tissue, apeptone, a yeast extract, a sugar such as dextrose or glucose, an NaCland/or Na₂PO₄ salt.

Still more particularly, said culture medium is a conventional mediumfor cultivating anaerobic bacteria such as so called broth media of theheart-brain type, Columbia media with 5% of sheep blood or a Schaedlermedium as described hereafter. Other suitable conventional media are theBrucella or Wilkins-Chagren media. Such acellular culture media are wellknown to one skilled in the art.

In particular it is possible to use polyvalent culture media foranaerobic microorganisms, notably Schaedler medium, said medium beingsupplemented with carbohydrate(s), preferably starch and sugar(s), andwith antioxidant compound(s).

The inventors actually tested different molecules having an antioxidantactivity and discovered that certain anti-oxidant compounds in certainconcentrations have a greater effect on the growth of said bacteria.

Other features and advantages of the present invention will becomebetter apparent upon reading the description which follows, made in anillustrative and non-limiting way, of an exemplary embodiment.

In order to illustrate the invention, the inventors cultivatedmethanogenic archaea notoriously recognized as strict anaerobic(recognized as being only cultivated in the strict absence of oxygen),in an aerobic atmosphere (i.e. open air, containing about 16% of oxygen)and in the presence of bacteria notoriously recognized as strictanaerobic bacteria, in bacterial and archae culture media notoriouslyrecognized as anaerobic, said culture media being supplemented withanti-oxidant compounds for supporting growth in an ambient airatmosphere and supplemented with carbohydrate(s) for producing H₂ andCH₄.

1) Used Strains.

A methanogenic archaea Methanobrevibacter smithii of strain DSM 861 wasobtained from the German collection of microorganisms and cell cultures(DSMZ, Braunschweig, Germany) also deposited at the ATCC under numberATCC 35061.

An archaea Methanomassiliicoccus luminyensis deposited at the depositcollection of microorganisms DSMZ (Germany), according to the BudapestTreaty, under number DSM 24529 was also tested as described in FR 124779 (published under number 2 990 954).

Moreover, a strain of the anaerobic bacterium Bacteroidesthetaiotaomicron was obtained through the <<culturomics>> study of theinventors (Lagier J C et al., Microbial culturomics: paradigm shift inthe human gut microbiome study. Clin Microbiol Infect. 2012; 18:1185-93)also accessible in diverse deposit collections (CSUR P766 also depositedaccording to the Budapest Treaty at the deposit collection ofmicro-organisms DSMZ (Germany) on May 19, 2014 under the number of DSM28808, other strains are also accessible in diverse deposit collectionssuch as the strains DSM 2079, ATCC 29148 and NCTC 10582).

Another following anaerobic bacterium strain was also tested:Lactococcus lactis deposited according to the Budapest Treaty at thedeposit collection of micro-organisms DSMZ (Germany) on May 19, 2014under the number of DSM 28809, also available in diverse depositcollections including the National Collection of micro-organism culturesde CNCM 1-2716.

2) Aerobic Culture of Anaerobic Microorganisms by Means of Antioxidants.

For their production in a sufficient amount, both strains M. smithii orM. luminyensis and B. thetaiotaomicron were cultivated in an anaerobicatmosphere at 37° C. in a polyvalent culture medium. The Schaedlermedium (Reference 42098; BioMérieux, La Balmes-les-Grottes, France) wasalso tested as well as the medium designated as <<SAB medium>> describedin FR 124 779 (published under the number of 2 990 954) and WO2013/0044933 customarily used for cultivating human methanogenic archaeaand which seems to be suitable also for the cultivation of anaerobicbacteria.

The Schaedler medium (marketed by BioMérieux, Marcy l′étoile, France)had the following composition for 1 litre:

Enzymatic digested material of casein 5.6 g   Enzymatic digestedmaterial of soya cake 1 g Enzymatic digested material of animal tissue 5g Yeast extract 5 g NaCl 1.7 g   Potassium phosphate 0.82 g   Dextrose5.82 g   Tris (hydroxymethyl) aminomethane 3 g Hemin 0.01 g   L-cysteine0.4 g  

The SAB medium used was without any Na₂S and of L-cysteine andcomprised: NiCl₂×6H₂O (0.07 mg/l), Na₂MoO₄×2H₂O (0.02 mg/l), FeSO₄×7H₂O(0.2 mg/l), MgSO₄×7H₂O (0.01 g/l), K₂HPO₄ (0.5 g/l), KH₂PO₄ (0.5 g/l),KCl (0.05 g/l), CaCl₂ (0.05 g/l), NaCl (1.5 g/l), NH₄Cl (1 g/l),Na-Acetate (1 g/l), yeast extract (1 g/l), Biotrypticase (1 g/l), Widdeltrace amount solution (2 ml/l), Balch trace element solution (10 ml/l),Resazurin (1 mg/l), Na₂Se₂O₃.5H₂O (0.015 mg/l), Na₂WO₄.2H₂O (0.02 mg/l),NiCl₂.6H₂O (0.07 mg/l), NaHCO₃ (4 g/l), Na-Formate (0.4 g/l), Methanol(40 mM), Balch medium vitamin solution (10 ml/l), valeric acid (0.6g/l), isovaleric acid (0.6 g/l), 2-methylbutyric acid (0.6 g/l),isobutyric acid (0.6 g/l), 2-methylvaleric acid (0.6 g/l).

The trace elements of Balch comprise NaCl, FeSO₄, ZnSO₄, MgSO₄, MnSO₄,CoSO₄, H₃BO₃, NiCl₂, Na₂MoO₄, CaCl₂.

The trace elements of Widdel comprise FeCl₂, CoCl₂, MnCl₂, ZnCl₂, H₃BO₃,Na₂MoO₄, NiCl₂ and CuCl₂.

The vitamins of the Balch medium comprise biotin, folic acid, pyridoxinehydrochloride, thiamine hydrochloride, riboflavin, nicotinic acid,DL-calcium pantothenate, vitamin B12, p-aminobenzoic acid and lipoicacid.

This Schaedler medium as well as this SAB medium were supplemented byadding carbohydrate(s) i.e. 1 g/L of rice starch and 1 g/L of glucose(Sigma-Aldrich, Saint-Quentin Fallavier, France) and addition ofanti-oxidant compounds i.e. supplemented with the addition of 1 g/L ofascorbic acid (VWR International, Louvain, Belgium), 0.1 g/L of uricacid and 0.1 g/L of glutathione (Sigma-Aldrich, Saint-Quentin Fallavier,France).

The resazurin is applied as an indicator of oxidation-reduction at aconcentration of 0.1 mg/mL for controlling the presence of oxygen(oxidized resazurin has a pink color and becomes transparent in theabsence of oxygen).

The aerobic culture in ambient air of M. smithii or M. luminyensis andB. thetaiotaomicron was carried out in separate containers and in a samecontainer incubated at 37° C. containing the culture medium supplementedwith the addition of anti-oxidant compounds and carbon source compounds.The pH was adjusted to 7.5 by adding 10M KOH.

Both strains were cultivated separately as well as in a co-culture,under aerobic conditions and by inoculation of 10⁵ organism/mL of eachstrain with the culture medium supplemented according to the presentinvention and in parallel with the Schaedler or SAB medium supplementedwith carbohydrates as mentioned above but, on the other hand without anyanti-oxidant compounds.

The positive controls consist in a tube containing the supplementedculture medium above, inoculated under anaerobic conditions with 10⁸microorganisms/L of M. smithii or M. luminyensis in the presence of agas mixture of 80% H₂+20% of CO₂ at the pressure of two atmospheresrequired for optimum growth of the methanogenic archaea. Thesupplemented culture medium inoculated under anaerobic conditions with10⁸ microorganisms/L of M. smithii or M. luminyensis without his gasmixture was introduced in order to check the growth of this methanogenicarchaea without H₂. The culture medium supplemented by adding 1 g/L ofrice starch and 1 g/L of glucose inoculated under anaerobic conditionswith 10⁸ cells/L of B. thetaiotaomicron was introduced as a positivecontrol and for checking the production of H₂ by B. thetaiotaomicron inan anaerobic culture. These controls were carried out in parallel in anambient atmosphere (aerobic). The non-inoculated culture medium wasintroduced as a negative control.

3) Detection of the Growth by Gas Chromatography.

The growth of M. smithii or M. luminyensis was daily evaluated by theproduction of methane and the growth of B. thetaiotaomicron was dailyevaluated by the production of hydrogen. The measurement of methane andof hydrogen was conducted by means of a gas chromatograph GC-8A(Shimadzu, Champs-sur-Marne, France) equipped with a heat conductivitydetector and a Chromosorb WAW 80/100 meshes column SP100 (Alltech,Carquefou, France). The nitrogen N₂ at a pressure of 100 kPa was used asa carrier gas. The detector and the temperatures of the injector were200° C. and the temperature of the column was 150° C.

4) Results.

4.1) Controls.

The negative controls remain negative without any growth occurring afterone week of incubation indicating that the results reported here are notsimply the result of a contamination by other microorganisms.

The positive controls were positive with production of methane observedin the anaerobic culture of M. stmithii or M. luminyensis and with aproduction of hydrogen observed in the anaerobic culture of B.thetaiotaomicron. There was no detectable production of methane in theculture of M. smithii or M. luminyensis inoculated alone under anaerobicconditions and under aerobic conditions without any gas mixture. Also,the culture of B. thetaiotaomicron inoculated under aerobic conditionswithout any antioxidant compounds remained negative and hydrogen was notproduced.

4.2) Aerobic Co-Culture.

After incubation for 24 hours at 37° C. in ambient air (under aerobicconditions), a culture medium without any anti-oxidant compoundsretained its pink color indicating the presence of oxygen. The aerobicculture medium with the anti-oxidant compounds became transparentindicating the absence of oxygen after its reduction by the antioxidantcompounds.

The culture medium inoculated under aerobic conditions with M. smithiior M. luminyensis with the anti-oxidant compounds became transparent,but the culture remained negative and methane was not produced. Theculture medium inoculated under aerobic conditions with B.thetaiotaomicron in the presence of the antioxidants became transparentand bacterial growth was observed with production of hydrogen.

The co-cultures of M. smithii or M. luminyensis and B. thetaiotaomicronachieved under aerobic conditions with anti-oxidant compounds all gave apositive culture for B. thetaiotaomicron with production of hydrogenafter 24 hour incubation. On the other hand, in certain experiments, nogrowth was observed for M. smithii or M. luminyensis (although theculture medium became transparent). The inventors made the assumptionthat M. smithii or M. luminyensis were dead because of their exposure tooxygen before the medium was reduced by the anti-oxidant compounds.

Experiments were conducted with the introduction of B. thetaiotaomicronat t0 and M. smithii or M. luminyensis added at t0+24 hours ofincubation. The addition of M. smithii or M. luminyensis after 24 hoursof incubation in every case gave the possibility of re-establishing itsgrowth by consumption of the hydrogen produced beforehand by B.thetaiotaomicron in the presence of antioxidant compounds and byavoiding mortal exposure to oxygen.

This same experiment was conducted without any antioxidant compound andthe culture remained negative for both of the tested strains.

On the other hand, the anaerobic bacterium Lactococcus lactis DSM 28809was also successfully tested combined with both of these archaea.

4.3) Interpretation

These results indicate that it is possible to cultivate in ambient air(aerobic condition) bacteria notoriously recognized as strictlyanaerobic, in a suitable medium containing a suitable mixture ofantioxidants.

Under these conditions, the anaerobic bacteria produce hydrogen whichmay then be used in a second phase by the methanogenic archaea forproducing methane.

It is shown that it is possible to produce methane in ambient air, undersuitable conditions of co-culture of bacteria and of anaerobicmethanogens in a culture medium supplemented with carbon sourcecompounds and anti-oxidant compounds.

The introduction of the methanogenic archaea in a medium alreadycontaining fermentation products and hydrogen is preferable.

1-15. (canceled)
 16. A method for producing methane gas, the methodcomprising: co-culturing, in a reactor under an aerobic atmosphere, atleast: a first microorganism that is an anaerobic bacterium capable toproduce hydrogen by fermentation in the presence of at least one of asubstrate and a culture medium comprising or supplemented with acarbohydrate, and a second microorganism that is a methanogenic Archaeacapable to produce methane from hydrogen and from at least one of asubstrate and a culture medium comprising, or supplemented with, acarbohydrate, and an organic and mineral substrate comprising componentsof culture media able to allow the cultivation of both said first andsaid second microorganisms, said culture medium comprising, or beingsupplemented with, a carbohydrate and further being supplemented with anantioxidant compound.
 17. The method according to claim 16, wherein thecultivation of said first microorganism is first carried out in saidsubstrate, and said second microorganism is introduced after said firstmicroorganism has produced fermentation products and hydrogen.
 18. Themethod according to claim 16, wherein said antioxidant compound isselected from the group consisting of ascorbic acid, uric acid, andglutathion.
 19. The method according to claim 16, wherein said secondmicroorganism is a genus selected from the group consisting ofMethanobrevibacter, Methanosphaera, Methanomassiliicoccus,Methanobacterium, Methanococcus, and Methanosaeta.
 20. The methodaccording to claim 16, wherein said second microorganism is selectedfrom the group consisting of Methanobrevibacter smithii,Methanobrevibacter oralis, Methanosphaera stadtmanae,Methanomassiliicoccus luminyensis, Methanobacterium beijingense, andMethanosaeta concilii.
 21. The method according to claim 16, whereinsaid second microorganism is Methanobrevibacter smithii, orMethanomassiliicoccus luminyensis.
 22. The method according to claim 16,wherein said first microorganism belongs to Bacteroidetes orActinobacteria families.
 23. The method according to claim 16, whereinsaid first microorgansism is a Bacteroides genus.
 24. The methodaccording to claim 16, wherein said first microorganism is Bacteroidesthetaiotaomicron, or Lactococcus lactis.
 25. The method according toclaim 16, wherein said first microorganism is Lactococcus lactis. 26.The method according to claim 16, wherein said second microorganism isMethanobrevibacter smithii DSM 861 or Methanomassiliicoccus luminyensisDSM 24529, and the first microorganism is Bacteroides thetaiotaomicronDSM 28808, or Lactococcus lactis DSM
 28809. 27. The method according toclaim 16, wherein said culture medium comprises: a plurality of carbonsources, a phosphorus source, a nitrogen source, and at least one saltof a metal selected from the group consisting of K, Mg, Na, and Ca. 28.The method according to claim 16, wherein said antioxidant compound isadded at a concentration of from 1 mg/l to 2 g/l.
 29. The methodaccording to claim 16, wherein said culture medium comprises apH-regulating buffer substance for adjusting the pH from 7 to 7.5. 30.The method according to claim 16, wherein said culture medium comprisescomponents of Schaedler's medium supplemented with a carbohydrate and asugar, and with said antioxidant compound.
 31. The method according toclaim 16, wherein the aerobic atmosphere is ambient air.